Nsp-interleukin-10 proteins and uses thereof

ABSTRACT

Disclosed herein are Nsp-IL10 polypeptides comprising an Nsp polypeptide and an IL10 polypeptide. In some embodiments, Nsp-IL10 polypeptide is capable of activating an NGF signaling pathway, an IL10 signaling pathway, or both. Also disclosed are methods for treating a disease comprising administering an Nsp-IL10 polypeptide. The methods include treating diseases associated with joint inflammation, such as osteoarthritis.

RELATED APPLICATIONS

This application claims priority to U.S. Application Ser. No. 62/775,433filed Dec. 5, 2018. U.S. Application Ser. No. 62/775,433 is incorporatedherein by reference in its entirety for all purposes.

FIELD

The disclosure generally relates to proteins, including fusion proteins,and methods for their use in treating diseases, particularlyinflammatory diseases. Specifically, the disclosed polypeptides containsequences from Nerve Growth Factor (NGF) and Interleukin-10.

BACKGROUND

Pain and loss of tissue function caused chronic inflammatory diseaseshas long been a major clinical challenge. For example, osteoarthritis(OA), the most prevalent degenerative joint disease worldwide, affectsup to 20% of the population in the U.S., and is the most common cause ofmobility loss, severely affecting the quality of life, workproductivity, cost of healthcare. There is no cure for OA, and currentclinical OA management is mainly concerned with symptom reduction, e.g.,pain, swelling, stiffness, with oral non-steroidal anti-inflammatorydrugs (NSAIDs) being the most commonly used pharmacological treatment atmid-stage of the disease, and arthroplasty, an irreversible procedure,as the final solution to maintain joint function. There are substantialgaps in the knowledge of the pathogenesis and effective interventionsfor early stage OA, which may prevent or delay disease development andmaintain proper joint functions.

Interleukin-10 (IL10) exhibits a range of physiological properties,including anti-cancer and anti-tumor properties as well as having rolesin inflammation. Dysregulation of IL10 is associated with autoimmunediseases and increased pathology in response to infection. Through avariety of mechanisms, IL10 produces anti-inflammatory responses whichserve to modulate immune responses.

Nerve Growth Factor (NGF) was originally identified (and thereforenamed) based on its functions in promoting neuronal survival anddifferentiation. However, recent studies show that NGF functions in anarray of biological processes. In particular, NGF has been implicated inthe transmission and maintenance of persistent or chronic pain andinflammation. However, mechanisms by which NGF and NGF-derivedpolypeptides bind surface receptors may influence the signaling pathwayand hence, overall response of activated cells.

While numerous factors are known to be involved in the control ofinflammatory responses, the network of molecular interactions are poorlyunderstood and thus, existing treatments are limited in their capacitiesto treat underlying causes of inflammation. The polypeptide compositionsand methods disclosed herein address these and other needs.

SUMMARY

The present invention relates to polypeptides containing amino acidsequences derived from Nerve Growth Factor (NGF) and Interleukin-10(IL-10; IL10) and methods for uses thereof. The present disclosureaddresses at least a portion of the problems in the prior art byproviding a polypeptide comprising an NGF polypeptide and an IL-10polypeptide which can be co-expressed and used to treat variousinflammatory conditions.

In one aspect, disclosed herein is an Nsp-IL10 polypeptide wherein Nspis a portion of an NGF polypeptide that binds to an NGF receptor. Insome embodiments, the Nsp polypeptide and the IL10 polypeptide aredirectly linked. In some embodiments, the polypeptide comprises a linkerbetween the Nsp polypeptide and the IL10 polypeptide.

In another aspect, provided herein are methods of treating a subjectwith a disease comprising administering to the subject an Nsp-IL10polypeptide comprising an Nsp polypeptide and an IL10 polypeptide. Insome embodiments, the disease is an inflammatory disease, for instance,joint inflammation (e.g., osteoarthritis). In some embodiments, themethod treats the disease by reducing inflammation, pain, tissuedegeneration, or combinations thereof.

In another aspect, provided herein are kits comprising a vectorcomprising a polynucleotide sequence encoding an Nsp-IL10 polynucleotideoperably linked to a gene promoter (referred to herein as promoter). TheNsp-IL10 polynucleotide comprises an Nsp polynucleotide and an IL10polynucleotide.

Additional aspects and advantages of the disclosure will be set forth,in part, in the detailed description and any claims which follow, and inpart will be derived from the detailed description or can be learned bypractice of the various aspects of the disclosure. The advantagesdescribed below will be realized and attained by means of the elementsand combinations particularly pointed out in the appended claims. It isto be understood that both the foregoing general description and thefollowing detailed description are for purposes of example, areexplanatory only, and are not restrictive of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate certain examples of the presentdisclosure and together with the description, serve to explain, withoutlimitation, the principles of the disclosure. Like numbers represent thesame element(s) throughout the figures.

FIGS. 1A to 1C are schematics showing Nsp-IL10 polypeptide expressionconstructs and resultant Nsp-IL10 polypeptides. FIG. 1A is a schematicshowing organization of genetic elements in some Nsp-IL10 polypeptideexpression constructs. FIG. 1B is a schematic showing predicted tertiarystructure of an Nsp-IL10 polypeptide expressed from construct b in FIG.1A. FIG. 1C is a schematic showing an Nsp-IL10 polypeptide bindingcell-surface receptors in NGFR+ cells, found for example in a jointcavity.

FIGS. 2A and 2B depict a strategy for expression of an Nsp-IL10polypeptide. FIG. 2A is a schematic showing an IL-10 expression plasmidusable for cloning Nsp-IL10 constructs. FIG. 2B is a schematic showingan Nsp-IL10 polypeptide construct and the results of a Western blotdemonstrating expression of the construct in HEK293 cells.

FIG. 3 is an immunoblot showing the downstream effects (expression ofP-TrkA, TrkA, SIRT1 and GAPDH) of Nsp-IL10 polypeptide expression inhealthy (left) and diseased (osteoarthritis; right) articularchondrocytes (AC) via Western blot analysis.

DETAILED DESCRIPTION

The following description of the disclosure is provided as an enablingteaching of the disclosure in its best, currently known embodiment(s).To this end, those skilled in the relevant art will recognize andappreciate that many changes can be made to the various embodiments ofthe invention described herein, while still obtaining the beneficialresults of the present disclosure. It will also be apparent that some ofthe desired benefits of the present disclosure can be obtained byselecting some of the features of the present disclosure withoututilizing other features. Accordingly, those who work in the art willrecognize that many modifications and adaptations to the presentdisclosure are possible and can even be desirable in certaincircumstances and are a part of the present disclosure. Thus, thefollowing description is provided as illustrative of the principles ofthe present disclosure and not in limitation thereof.

Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. The following definitions areprovided for the full understanding of terms used in this specification.

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular polypeptide is disclosed and discussed and anumber of modifications that can be made to the polypeptide arediscussed, specifically contemplated is each and every combination andpermutation of the polypeptide and the modifications that are possibleunless specifically indicated to the contrary. Thus, if a class ofpolypeptides A, B, and C are disclosed as well as a class ofpolypeptides D, E, and F and an example of a combination polypeptide,or, for example, a combination polypeptide comprising A-D is disclosed,then even if each is not individually recited each is individually andcollectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F,C-D, C-E, and C-F are considered disclosed. Likewise, any subset orcombination of these is also disclosed. Thus, for example, the sub-groupof A-E, B-F, and C-E would be considered disclosed. This concept appliesto all aspects of this application including, but not limited to, stepsin methods of making and using the disclosed compositions. Thus, ifthere are a variety of additional steps that can be performed it isunderstood that each of these additional steps can be performed with anyspecific embodiment or combination of embodiments of the disclosedmethods.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures which can perform the same function which arerelated to the disclosed structures, and that these structures willultimately achieve the same result.

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; and the number ortype of embodiments described in the specification.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed.

As used in the specification and claims, the singular form “a,” “an,”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “an agent” includes a plurality ofagents, including mixtures thereof.

As used herein, the terms “may,” “optionally,” and “may optionally” areused interchangeably and are meant to include cases in which thecondition occurs as well as cases in which the condition does not occur.Thus, for example, the statement that a formulation “may include anexcipient” is meant to include cases in which the formulation includesan excipient as well as cases in which the formulation does not includean excipient.

“Administration” to a subject includes any route of introducing ordelivering to a subject an agent. Administration can be carried out byany suitable route, including oral, topical, intravenous, subcutaneous,transcutaneous, transdermal, intramuscular, intra joint, parenteral,intra-arteriole, intradermal, intraventricular, intracranial,intraperitoneal, intralesional, intranasal, rectal, vaginal, byinhalation, via an implanted reservoir, parenteral (e.g., subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional,and intracranial injections or infusion techniques), and the like.“Concurrent administration”, “administration in combination”,“simultaneous administration” or “administered simultaneously” as usedherein, means that the compounds are administered at the same point intime, overlapping in time, or essentially immediately following oneanother. In the latter case, the two compounds are administered at timessufficiently close that the results observed are indistinguishable fromthose achieved when the compounds are administered at the same point intime. “Systemic administration” refers to the introducing or deliveringto a subject an agent via a route which introduces or delivers the agentto extensive areas of the subject's body (e.g. greater than 50% of thebody), for example through entrance into the circulatory or lymphsystems. By contrast, “local administration” refers to the introducingor delivery to a subject an agent via a route which introduces ordelivers the agent to the area or area immediately adjacent to the pointof administration and does not introduce the agent systemically in atherapeutically significant amount. For example, locally administeredagents are easily detectable in the local vicinity of the point ofadministration, but are undetectable or detectable at negligible amountsin distal parts of the subject's body. Administration includesself-administration and the administration by another.

“Codon optimized” as it refers to genes or coding regions of nucleicacid molecules for the transformation of various hosts, refers to thealteration of codons in the gene or coding regions of polynucleic acidmolecules to reflect the typical codon usage of a selected organismwithout altering the polypeptide encoded by the DNA. Due to redundancyin the genetic code, multiple codons can encode the same amino acid.Some organisms have a preference for using a particular codon to encodea particular amino acid, as determined by the percentage in which thatparticular amino acid is encoded by that particular codon throughout theorganism's genome. Such optimization includes replacing at least one, ormore than one, or a significant number, of codons with one or morecodons that are more frequently used in the genes of that selectedorganism.

“Gene expression” and “protein expression” refer to the process by whichpolynucleotides are transcribed into mRNA and the process by which thetranscribed mRNA is subsequently being translated into peptides,polypeptides, or proteins, respectively. If the polynucleotide isderived from genomic DNA, expression may include splicing of the mRNA ina eukaryotic cell.

“Identical” or percent “identity,” in the context of two or more nucleicacids or polypeptide sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage of aminoacid residues or nucleotides that are the same (e.g., about 60%identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or higher identity over a specified region when compared and alignedfor maximum correspondence over a comparison window or designatedregion) as measured using a BLAST or BLAST 2.0 sequence comparisonalgorithms with default parameters described below, or by manualalignment and visual inspection (see, e.g., NCBI web site or the like).Such sequences are then said to be “substantially identical.” Thisdefinition also refers to, or may be applied to, the complement of atest sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about10 amino acids or 20 nucleotides in length, or more preferably over aregion that is 10-50 amino acids or 20-50 nucleotides in length. As usedherein, percent (%) amino acid sequence identity is defined as thepercentage of amino acids in a candidate sequence that are identical tothe amino acids in a reference sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity. Alignment for purposes of determining percentsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)software. Appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full-length ofthe sequences being compared can be determined by known methods.

For sequence comparisons, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410).These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word lengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01.

The “linker” used herein refers to at least a bivalent moiety with asite of attachment for a first polypeptide and a site of attachment fora second polypeptide. For example, the first polypeptide or the secondpolypeptide can be attached to the linker at its N-terminus, itsC-terminus or via a functional group on one of the side chains. Thelinker is sufficient to separate the first and the second polypeptidesby at least one amino acid and in some embodiments by more than oneamino acid. In some embodiments, the linker is sufficiently flexible toallow the first polypeptide to bind target molecules in a manner whichis independent of the second polypeptide. In some embodiments, thelinker is sufficiently flexible to allow the second polypeptide to bindtarget molecules in a manner which is independent of the firstpolypeptide. In some embodiments, the first polypeptide is an Nsppolypeptide and the second polypeptide is an IL-10 polypeptide. In someembodiments, the first polypeptide is an IL-10 polypeptide and thesecond polypeptide is an Nsp polypeptide.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are near each other, and, inthe case of a secretory leader, contiguous and in reading phase.However, operably linked nucleic acids (e.g. enhancers and codingsequences) do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice. In some embodiments, a promoter is operablylinked with a coding sequence when it is capable of affecting (e.g.modulating relative to the absence of the promoter) the expression of aprotein from that coding sequence (e.g., the coding sequence is underthe transcriptional control of the promoter).

“Pharmaceutically acceptable” component can refer to a component that isnot biologically or otherwise undesirable, e.g., the component may beincorporated into a pharmaceutical formulation of the invention andadministered to a subject as described herein without causingsignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of the formulationin which it is contained. When used in reference to administration to ahuman, the term generally implies the component has met the requiredstandards of toxicological and manufacturing testing or that it isincluded on the Inactive Ingredient Guide prepared by the U.S. Food andDrug Administration.

“Pharmaceutically acceptable carrier” (sometimes referred to as a“carrier”) means a carrier or excipient that is useful in preparing apharmaceutical or therapeutic composition that is generally safe andnon-toxic, and includes a carrier that is acceptable for veterinaryand/or human pharmaceutical or therapeutic use. The terms “carrier” or“pharmaceutically acceptable carrier” can include, but are not limitedto, phosphate buffered saline solution, water, emulsions (such as anoil/water or water/oil emulsion) and/or various types of wetting agents.As used herein, the term “carrier” encompasses, but is not limited to,any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer,lipid, stabilizer, or other material well known in the art for use inpharmaceutical formulations and as described further herein.

“Polynucleotide” and “oligonucleotide” are used interchangeably, andrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: a gene or gene fragment, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. The sequence of nucleotides maybe interrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. A polynucleotide is composed of a specific sequenceof four nucleotide bases: adenine (A); cytosine (C); guanine (G);thymine (T); and uracil (U) for thymine (T) when the polynucleotide isRNA. Thus, the term “polynucleotide sequence” is the alphabeticalrepresentation of a polynucleotide molecule.

“Polypeptide” is used in its broadest sense to refer to a compound oftwo or more subunit amino acids, amino acid analogs, or peptidomimetics.The subunits may be linked by peptide bonds. In another embodiment, thesubunit may be linked by other bonds, e.g. ester, ether, etc. As usedherein the term “amino acid” refers to either natural and/or unnaturalor synthetic amino acids, including glycine and both the D or L opticalisomers, and amino acid analogs and peptidomimetics.

“Specifically binds” when referring to a polypeptide (includingantibodies) or receptor, refers to a binding reaction which isdeterminative of the presence of the protein or polypeptide or receptorin a heterogeneous population of proteins and other biologics. Thus,under designated conditions (e.g. immunoassay conditions in the case ofan antibody), a specified ligand or antibody “specifically binds” to itsparticular “target” (e.g. an antibody specifically binds to anendothelial antigen) when it does not bind in a significant amount toother proteins present in the sample or to other proteins to which theligand or antibody may come in contact in an organism. Generally, afirst molecule that “specifically binds” a second molecule has anaffinity constant (Ka) greater than about 10⁵ M⁻¹ (e.g., 10⁶ M⁻¹, 10⁷M⁻¹, 10⁸ M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, and 10¹² M⁻¹ or more) withthat second molecule.

“Therapeutic agent” refers to any composition that has a beneficialbiological effect. Beneficial biological effects include boththerapeutic effects, e.g., treatment of a disorder or other undesirablephysiological condition, and prophylactic effects, e.g., preventingsymptoms of a disorder or other undesirable physiological condition(e.g., rheumatoid arthritis). The terms also encompass pharmaceuticallyacceptable, pharmacologically active derivatives of beneficial agentsspecifically mentioned herein, including, but not limited to, salts,esters, amides, proagents, active metabolites, isomers, fragments,analogs, and the like. When the terms “therapeutic agent” is used, then,or when a particular agent is specifically identified, it is to beunderstood that the term includes the agent per se as well aspharmaceutically acceptable, pharmacologically active salts, esters,amides, proagents, conjugates, active metabolites, isomers, fragments,analogs, etc.

“Therapeutically effective amount” or “therapeutically effective dose”of a composition (e.g. a composition comprising an agent) refers to anamount that is effective to achieve a desired therapeutic result. Insome embodiments, a desired therapeutic result is the control of chronicinflammation. Therapeutically effective amounts of a given therapeuticagent will typically vary with respect to factors such as the type andseverity of the disorder or disease being treated and the age, gender,weight, and general condition of the subject. Thus, it is not alwayspossible to specify a quantified “therapeutically effective amount.”However, an appropriate “therapeutically effective amount” in anysubject case may be determined by one of ordinary skill in the art usingroutine experimentation. The term can also refer to an amount of atherapeutic agent, or a rate of delivery of a therapeutic agent (e.g.,amount over time), effective to facilitate a desired therapeutic effect,such as pain relief. The precise desired therapeutic effect will varyaccording to the condition to be treated, the tolerance of the subject,the agent and/or agent formulation to be administered (e.g., the potencyof the therapeutic agent, the concentration of agent in the formulation,and the like), and a variety of other factors that are appreciated bythose of ordinary skill in the art. It is understood that, unlessspecifically stated otherwise, a “therapeutically effective amount” of atherapeutic agent can also refer to an amount that is a prophylacticallyeffective amount. In some instances, a desired biological or medicalresponse is achieved following administration of multiple dosages of thecomposition to the subject over a period of days, weeks, or years.

As used herein, “transgene” refers to exogenous genetic material (e.g.,one or more polynucleotides) that has been or can be artificiallyprovided to a cell. The term can be used to refer to a “recombinant”polynucleotide encoding any of the herein disclosed polypeptides thatare the subject of the present disclosure. The term “recombinant” refersto a sequence (e.g., polynucleotide or polypeptide sequence) which doesnot occur in the cell to be artificially provided with the sequence, oris linked to another polynucleotide in an arrangement which does notoccur in the cell to be artificially provided with the sequence. It isunderstood that “artificial” refers to non-natural occurrence in thehost cell and includes manipulation by man, machine, exogenous factors(e.g., enzymes, viruses, etc.), other non-natural manipulations, orcombinations thereof. A transgene can comprise a gene operably linked toa promoter (e.g., an open reading frame), although is not limitedthereto. Upon artificially providing a transgene to a cell, thetransgene may integrate into the host cell chromosome, existextrachromosomally, or exist in any combination thereof.

“Treat”, “treating”, “treatment” and grammatical variations thereof, insome instances include partially or completely reducing the severity ofinflammation, reducing the overall area affected by inflammation, andreducing the duration of inflammation as compared with prior totreatment of the subject or as compared with the incidence of suchsymptom in a general or study population. “Treat”, “treating”,“treatment” and grammatical variations thereof, in some or furtherinstances include partially or completely reducing the severity ofarthritis (e.g., osteoarthritis), reducing the overall area affected byarthritis, and reducing the duration of arthritis as compared with priorto treatment of the subject or as compared with the incidence of suchsymptom in a general or study population.

“Vector” means a DNA construct containing a DNA sequence which isoperably linked to a suitable control sequence capable of effecting theexpression of the DNA in a suitable host. Such control sequences includea promoter to effect transcription, an optional operator sequence tocontrol such transcription, a sequence encoding suitable mRNA ribosomebinding sites, and sequences which control the termination oftranscription and translation. The vector may be a plasmid, a phageparticle, or simply a potential genomic insert. Once transformed into asuitable host, the vector may replicate and function independently ofthe host genome, or may in some instances, integrate into the genomeitself. A plasmid is the most commonly used form of a vector; however,the invention is intended to include such other forms of vectors whichserve equivalent function as and which are, or become, known in the art.

Nsp-IL10 Polypeptides

It should be understood that the Nsp-IL10 polypeptides of the presentdisclosure can be used in combination with the various compositions,methods, products, and applications disclosed herein.

In one aspect, disclosed herein are Nsp-IL10 polypeptides comprising anNsp polypeptide and an IL10 polypeptide. A surprising discovery of thepresent invention is that these Nsp-IL10 polypeptides can coordinatelyor simultaneously activate both NGF and IL-10 signaling pathways. TheNsp-IL10 polypeptide, when administered to a subject, can treat chronicinflammatory diseases such as osteoarthritis. In some embodiments, theNsp-IL10 polypeptide maintains tissue homeostasis and/or enhances immunemodulation at least by reducing inflammation, pain, and/or delayingtissue degeneration. The herein disclosed Nsp-IL10 polypeptides are, insome embodiments, therefore capable of treating the underlying causes ofchronic inflammatory diseases rather than simply reducing orameliorating symptoms of such diseases.

As used herein, the term “Nsp” refers to an NGF small protein, or aportion of an NGF polypeptide that binds an NGF receptor (also called“NGFR”). “NGF” refers to a Nerve Growth Factor (NGF) polypeptide alsoknown as NGFβ and, in humans, is encoded by the NGF gene. In someembodiments, the NGF polypeptide or polynucleotide is that identified inone or more publicly available databases as follows: HGNC: 7808, EntrezGene: 4803, Ensembl: ENSG00000134259, OMIM: 162030, and UniProtKB:P01138. In some embodiments, the NGF polypeptide or polynucleotidecomprises the sequence of SEQ ID NO: 1, or a polypeptide sequence havingat or greater than about 80%, at or greater than about 85%, at orgreater than about 90%, at or greater than about 95%, or at or greaterthan about 98% homology with SEQ ID NO: 1, or a fragment thereof. TheNGF protein can be from any vertebrate, particularly from any mammalsuch as livestock such as cows, pigs, and sheep, primates such ashumans, gorillas and monkeys, rodents such as mice, rats and guineapigs, and other mammals such as horse, dog, bear, deer, dolphin,felines, etc. In some embodiments, the Nsp polypeptide is a portion ofhuman NGF.

The Nsp polypeptide comprises a portion of an NGF polypeptide that bindsan NGF receptor. The Nsp polypeptide can comprise more than one portionof NGF (e.g. an N-terminal portion and a C-terminal portion). In someembodiments, the Nsp polypeptide comprises the N-terminal half of NGF.Optionally, the Nsp polypeptide comprises the unstructured N-terminaldomain of NGF. By “unstructured,” it is meant the N-terminal domainlacks substantial alpha-helical or β-strand structure, and comprisesprimarily flexible loops with large degrees of freedom. In someembodiments, the Nsp polypeptide comprises amino acids 1-14 of NGF. Inother embodiments, the Nsp polypeptide comprises amino acids 1-12, 1-13,2-15, 3-16 or 4-17 of NGF.

In some embodiments, the Nsp polypeptide contains at least 60% (forexample, at least 60%, at least 65%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%) identity to SEQ IDNO: 2. In some embodiments, the Nsp polypeptide comprises the amino acidsequence of SEQ ID NO: 2.

In some embodiments, a polynucleotide encoding the Nsp polypeptidecomprises a nucleic acid sequence which is at least 60%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% identical to SEQ ID NO: 9. In some embodiments, apolynucleotide encoding the Nsp polypeptide comprises SEQ ID NO: 9.

NGF is a polypeptide known to bind at least two receptors accessible atthe outer surface of cell membranes: TrkA and p75NTR. In someembodiments, the NGF receptor is selected from a tyrosine kinasemembrane receptor (Trk) and p75NTR. In some embodiments, the NGFreceptor comprises Tyrosine kinase membrane Receptor A (TrkA).Accordingly, in some embodiments, the Nsp-IL10 polypeptide binds to TrkAor p75NTR. In some embodiments, the Nsp-IL10 polypeptide selectivelybinds TrkA (e.g., does not bind p75NTR). In some embodiments, theNsp-IL10 polypeptide preferably binds TrkA as compared to p75NTR. Insome embodiments, the Nsp-IL10 binding is agonistic. In otherembodiments, the Nsp-IL10 binding is antagonistic. In some embodiments,the Nsp-IL10 polypeptide is capable of selectively binding TrkA withoutsubstantially activating the p75NTR-mediated apoptotic pathway. In someembodiments, binding of the Nsp-IL10 polypeptide to an NGF receptoractivates an NGF signaling pathway. In some embodiments, binding of theNsp-IL10 polypeptide to an NGF receptor facilitates (e.g., promotes)dimerization of the NGF receptor. In some embodiments, binding of theNsp-IL10 polypeptide to an NGF receptor facilitates (e.g., promotes,increases) phosphorylation of the NGF receptor. In some embodiments, thephosphorylation comprises autophosphorylation. For example, in someembodiments, binding of NGF to TrkA results in dimerization andphosphorylation of TrkA, thereby initiating the NGF signaling pathway.

The NGF signaling pathway comprises several different signalingmediators, each having the potential to slightly or significantly alterthe cellular response to binding of NGF or the Nsp polypeptide to an NGFreceptor. For example, NGF-p75NTR binding can result in a signalingpathway which triggers apoptosis. Alternatively, NGF-TrkA binding canresult in a signaling pathway which activates a mitogen activatedprotein kinase (MAPK; also known as extracellular signal-regulatedkinase ERK) via ERK1/2 proteins. Alternatively, NGF-TrkA binding canresult in a signaling pathway which activates a phosphoinositide3-kinase. The phosphoinositide 3-kinase PI3K can phosphorylate andactivate protein kinase B (PKB; also known as AKT). Activation ofPI3K/AKT can result in cell protection, survival, and/or proliferation.

In some embodiments, the NGF signaling pathway comprises PI3K/AKTactivation. In some or further embodiments, the NGF signaling pathwayavoids p75NTR-mediated apoptosis, MAPK activation (ERK1/2 activation),or both. Thus, in some embodiments, the NGF signaling pathway includesonly PI3K/AKT activation. In some embodiments, the Nsp-IL10 polypeptideselectively binds TrkA and selectively activates signaling via thePI3K/AKT pathway.

The Nsp-IL10 polypeptide disclosed herein further comprises an IL10polypeptide. As used herein, “IL10” refers to Interleukin-10 or IL-10,an immune modulating cytokine. In some embodiments, the IL10 polypeptideor polynucleotide is that identified in one or more publicly availabledatabases as follows: HGNC: 5962, Entrez Gene: 3586, Ensembl:ENSG00000136634, OMIM: 124092, and UniProtKB: P22301. In someembodiments, the IL10 polypeptide or polynucleotide comprises thesequence of SEQ ID NO: 3, or a polypeptide sequence having at or greaterthan about 80%, at or greater than about 85%, at or greater than about90%, at or greater than about 95%, or at or greater than about 98%homology with SEQ ID NO: 3, or a fragment thereof.

As used herein, the term “IL10 polypeptide” refers to a polypeptide thatcomprises at least a portion of IL10, which portion binds an IL10receptor. In some embodiments, the IL10 polypeptide comprises afull-length IL10 including a signal peptide. The polypeptide of SEQ IDNO: 5 is an exemplary signal peptide. In other embodiments, the IL10polypeptide comprises a secreted form of IL10 (lacking a signalpeptide). In some embodiments, the IL10 polypeptide is from anyvertebrate, particularly from any mammal such as livestock such as cows,pigs, and sheep, primates such as humans, gorillas and monkeys, rodentssuch as mice, rats and guinea pigs, and other mammals such as horse,dog, bear, deer, dolphin, felines, etc.

In some embodiments, the IL10 polypeptide contains at least 60% (forexample, at least 60%, at least 65%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%) identity to theamino acid sequence of SEQ ID NO: 4. In some embodiments, the IL10polypeptide comprises the amino acid sequence of SEQ ID NO: 4.

In some embodiments, a polynucleotide encoding the IL10 polypeptidecomprises a nucleic acid sequence which is at least 60%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% identical to SEQ ID NO: 11. In some embodiments, apolynucleotide encoding the IL10 polypeptide comprises SEQ ID NO: 11.

In some embodiments, binding of the Nsp-IL10 peptide to an IL-10receptor (IL-10R) activates an IL10 signaling pathway. The IL-10receptor (IL-10R) can be tetrameric, being comprised of intracellulardomains which bind JAK1 and TYK2 kinases. Subsequent autophosphorylationof these kinases activates Signal Transducer and Activator ofTranscription (STAT) family proteins including STAT1 and STATS andprimarily including STAT3. STAT activation leads to transcriptionalregulation of an array of genes.

In some embodiments, Nsp-IL10 binding facilitates (e.g., promotes,increases) phosphorylation of IL-10R. In some embodiments, thephosphorylation comprises autophosphorylation. For example, binding ofNsp-IL10 to IL-10R results in phosphorylation of at least one of JAK1and TYK2, thereby initiating the IL10 signaling pathway. Optionally, theIL10 signaling pathway comprises transcriptional inhibition of cytokineexpression, for example, of pro-inflammatory cytokines. In someembodiments, the IL10 signaling pathway reduces expression of a cytokineselected from IFN-γ, IL-2, IL-3, TNFα and GM-CSF.

In some or further embodiments, the IL10 signaling pathway comprisesincreased expression of a deacetylase protein. Optionally, thedeacetylase is NAD-dependent. In some or further embodiments, the IL10signaling pathway comprises increased expression of a tissue agingmarker. Optionally, the tissue aging marker is a sirtuin family protein(e.g., SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7). Optionally,the tissue aging marker is Sirtuin1 (SIRT1).

Without wishing to be bound by any one particular mechanism, it isthought that the Nsp polypeptide in the Nsp-IL10 polypeptide functionsto bind NGFR, thereby delivering the IL-10 polypeptide to NGFR+ cells.In some embodiments, binding of the Nsp polypeptide to NGFR activates anNGFR signaling pathway, while the IL-10 polypeptide activates an IL-10signaling pathway in the same cell. In some embodiments, the Nsp-IL10polypeptide activates the NGFR signaling pathway in one cell, andactivates the IL-10 signaling pathway in a separate, adjacent or nearbycell.

The Nsp polypeptide and the IL10 polypeptide can be arranged within theNsp-IL10 polypeptide in a number of ways. The Nsp polypeptide and theIL10 polypeptide can be expressed from separate genetic constructs.Alternatively, the Nsp polypeptide and the IL10 polypeptide can beexpressed from the same genetic construct, for example as a singletranscript.

In some embodiments, the Nsp polypeptide and the IL10 polypeptide aredirectly linked. By “directly linked,” it is meant that the twopolypeptides are covalently attached in a single macromolecule, wherethere are no intervening amino acids between the different polypeptidesor where the individual polypeptides are connected to one another viaone or more intervening amino acids (e.g., linkers). In someembodiments, the Nsp and IL10 polypeptides are directly linked in asingle polypeptide, for example as a fusion protein comprising the Nspand IL10 polypeptides. In some embodiments, the Nsp and IL10polypeptides are directly linked by a post-translational modification,for example by a disulfide bridge (e.g., cysteine-cysteine disulfidebond).

In some embodiments, an Nsp polypeptide is directly linked to theN-terminal end of an IL10 polypeptide. In some embodiments, the Nsppolypeptide is directly linked to the C-terminal end of the IL10polypeptide. In some embodiments, the continuous Nsp polypeptide isinserted within the sequence of an IL10 polypeptide, wherein the IL10polypeptide includes an IL10 signal sequence. In some embodiments, theNsp polypeptide is inserted within the sequence of the IL10 polypeptideC-terminal to the IL10 signal peptide but N-terminal to the IL10 mature,secreted polypeptide. In some embodiments, the Nsp polypeptide isinserted between the N-terminal 18^(th) and 19^(th) amino acids of anIL10 polypeptide which includes an IL10 signal sequence. In otherembodiments, the Nsp polypeptide is directly linked to an IL10polypeptide that does not comprise a signal peptide.

In some embodiments, the Nsp-IL10 polypeptide further comprises alinker. For example, the Nsp polypeptide may be linked to the IL10polypeptide by an intervening linker comprising one or more amino acids.The linker can contain one, two, three, four, five, six, seven, eight,nine, ten, or a plurality of amino acids. The Nsp-IL10 polypeptide cancomprise more than one linkers (e.g., one, two, three, four, five, six,seven, eight, nine, ten, or a plurality of linkers).

In some embodiments, a linker is between the Nsp polypeptide and theIL10 polypeptide. In some embodiments, the linker is positioned betweenan N-terminal Nsp polypeptide and a C-terminal IL10 polypeptide.Alternatively, the linker is positioned between a C-terminal Nsppolypeptide and an N-terminal IL10 polypeptide. In some embodiments, thecontinuous Nsp polypeptide is inserted within the sequence of the IL10polypeptide, wherein a linker is positioned between the Nsp polypeptideand the IL10 polypeptide. In some embodiments, the Nsp polypeptide isinserted within the sequence of the IL10 polypeptide C-terminal to anIL10 signal peptide and N-terminal to an IL10 polypeptide, wherein alinker is positioned between the Nsp polypeptide and the IL10polypeptide. In some embodiments, the Nsp polypeptide is insertedbetween the N-terminal 18^(th) and 19^(th) amino acids of the IL10polypeptide, wherein a linker is positioned between the Nsp polypeptideand the IL10 polypeptide. In some embodiments, the polypeptide comprisesan Nsp polypeptide flanked by two linkers inserted within the sequenceof an IL10 polypeptide.

In some embodiments, the linker contains at least 60% (for example, atleast 60%, at least 65%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 99%) identity to the amino acidsequence of SEQ ID NO: 6. In some embodiments, the linker comprises theamino acid sequence of SEQ ID NO: 6.

In some embodiments, a polynucleotide encoding the linker comprises anucleic acid sequence which is at least 60%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to SEQ ID NO: 13. In some embodiments, a polynucleotideencoding the linker comprises SEQ ID NO: 13.

The Nsp-IL10 polypeptide can contain additional amino acid sequenceswhich are not involved in activating either the NGF pathway or the IL10pathway. For example, the Nsp-IL10 polypeptide can contain a signalpeptide for export of the polypeptide from a biological cell. The signalpeptide can be from a neurotrophin (e.g., NGF), IL10, or anotherexported protein. As another non-limiting example, the Nsp-IL10polypeptide can contain additional sequences for affinity-basedpurification (e.g., Myc-DDK) and/or post-translational modifications(e.g., cysteines for forming disulfide bonds).

In some embodiments, the Nsp-IL10 polypeptide contains at least 60% (forexample, at least 60%, at least 65%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%) identity to theamino acid sequence of SEQ ID NO: 7. In some embodiments, the Nsp-IL10polypeptide comprises the amino acid sequence of SEQ ID NO: 7.

In some embodiments, a polynucleotide encoding the Nsp-IL10 polypeptidecomprises a nucleic acid sequence which is at least 60%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% identical to SEQ ID NO: 14. In some embodiments, apolynucleotide encoding the Nsp-IL10 polypeptide comprises SEQ ID NO:14.

Functional IL10 is often in the form of a dimer. As such, the Nsp-IL10polypeptide can comprise Nsp-IL10 polypeptide homodimers. Alternatively,the Nsp-IL10 polypeptide can comprise an IL10 polypeptide and Nsp-IL10polypeptide heterodimer. As an example, FIG. 1B is a predicted structureof Nsp-IL10 polypeptide heterodimerized with IL10. In some embodiments,the Nsp-IL10 polypeptide can comprise a mixture of Nsp-IL10 polypeptidehomodimers and heterodimers comprising IL10 polypeptide and Nsp-IL10polypeptide.

The Nsp-IL10 polypeptide optionally comprises additional components suchas amino acid sequences (e.g., sequences of other proteins, linkersequences, non-proteinogenic amino acids, etc.) and other protein-boundmolecules (e.g., cofactors, small molecules, lipids, carbohydrates,nucleic acids, post-translational modifications such as acylation,glycosylation, hydroxylation, iodination, carbonylation, pegylation,etc.).

Also disclosed herein is a biological cell comprising an Nsp-IL10polypeptide comprising an Nsp polypeptide and an IL10 polypeptide. Forexample, a host cell (e.g., E. coli, mammalian cells) can be used forproduction of the Nsp-IL10 polypeptide. Alternatively, the biologicalcell can be bound by an Nsp-IL10 polypeptide. For example, a biologicalcell in a cell culture, tissue culture, or in a subject can be bound byan Nsp-IL10 polypeptide via a cell-membrane receptor (e.g., NGFR,IL10R). The biological cell bound by an Nsp-IL10 polypeptide can be invarious states of activation. For example, the biological cell may bebound but be non-activated for both NGF and IL10 pathways, bound andactivated for either NGF or IL10 pathway but not both, or be activatedfor both NGF and IL10 pathways.

Also disclosed herein is a composition comprising an Nsp-IL10polypeptide comprising an Nsp polypeptide and an IL10 polypeptide, and apharmaceutically acceptable excipient. Suitable excipients include, butare not limited to, salts, diluents, binders, fillers, solubilizers,disintegrants, preservatives, sorbents, and other components. Alsodisclosed herein is a medicament comprising a pharmaceutically effectiveamount of an Nsp-IL10 polypeptide comprising an Nsp polypeptide and anIL10 polypeptide. As an example, a pharmaceutically effective amount ofNsp-IL10 polypeptide can be formulated in a hydrogel, particularly aphotocrosslinkable and biodegradable hydrogel scaffold.

Methods of Treating

Also disclosed herein are methods of treating a subject with a diseasecomprising administering to the subject an Nsp-IL10 polypeptidecomprising an Nsp polypeptide and an IL10 polypeptide or an Nsp-IL10polynucleotide comprising an Nsp polynucleotide and an IL10polynucleotide. The Nsp-IL10 polypeptide and polynucleotide can be anyherein disclosed.

In some embodiments, the administering step can include any method ofintroducing the Nsp-IL10 polypeptide into the subject appropriate forthe polypeptide formulation. The administering step can include at leastone, two, three, four, five, six, seven, eight, nine, or at least tendosages. The administering step can be performed before the subjectexhibits disease symptoms (e.g., prophylactically), or during or afterdisease symptoms occur. The administering step can be performed priorto, concurrent with, or subsequent to administration of other agents tothe subject. The administering step can be performed with or withoutco-administration of additional agents (e.g., immunosuppressive agents,additional anti-inflammation agents).

The administering step can comprise administering the Nsp-IL10polypeptide as a purified polypeptide composition or in a cellularextract. In other embodiments, the administering step comprisesadministering a cell comprising a polynucleotide sequence encoding anNsp-IL10 polynucleotide operably linked to a promoter, wherein theNsp-IL10 polynucleotide comprises an Nsp polynucleotide and an IL10polynucleotide, and expressing the polypeptide from the polynucleotide.In some embodiments, the cell is a chondrocyte. In some embodiments, theadministering step comprises administering a polynucleotide sequenceencoding an Nsp-IL10 polynucleotide operably linked to a promoter,wherein the Nsp-IL10 polynucleotide comprises an Nsp polynucleotide andan IL10 polynucleotide, and expressing the polypeptide from thepolynucleotide. In some embodiments, the Nsp-IL10 polypeptide isexpressed by a virus. Unless specifically stated otherwise,administering a polypeptide, as used herein, includes administering apolypeptide (e.g., in purified or extract form), administering apolynucleotide which encodes the polypeptide (e.g., a transgene), andadministering both a polypeptide and a polynucleotide which encodes thepolypeptide. Unless specifically stated otherwise, administering apolynucleotide, as used herein, includes administering a polynucleotidewhich encodes a polypeptide, and administering both a polypeptide and apolynucleotide which encodes the polypeptide.

The subject can be any mammalian subject, for example a human, dog, cow,horse, mouse, rabbit, etc. In some embodiments, the subject is aprimate, particularly a human. The subject can be a male or female ofany age, race, creed, ethnicity, socio-economic status, or other generalclassifiers.

The disease can be any disease in which administration of an Nsp-IL10polypeptide can be used to treat. In some embodiments, the disease is aninflammatory disease. In some embodiments, the disease is chronicinflammation. Non-limiting examples of inflammatory diseases includejoint inflammation (e.g., osteoarthritis), rheumatoid arthritis,collagen antibody-induced arthritis, asthma, chronic peptic ulcer,tuberculosis, periodontitis, ulcerative colitis, Crohn's disease,sinusitis, hepatitis, bronchitis, appendicitis, dermatitis, meningitis,ankylosing spondylitis, celiac disease, idiopathic pulmonary fibrosis,lupus, systemic lupus erythematosus, psoriasis, type 1 diabetes,Addison's disease, allergy, arthritis, prostatitis, diverticulitis,glomerulonephritis, hidradenitis suppurativa, inflammatory boweldisease, interstitial cystitis, mast cell activation syndrome,mastocytosis, otitis, pelvic inflammatory disease, reperfusion injury,rheumatic fever, rhinitis, sarcoidosis, transplant rejection,vasculitis, atherosclerosis, gout, pleurisy, eczema, gastritis,splenitis, laryngitis, thyroiditis, pharyngitis, multiple sclerosis,myopathies, seborrheic dermatitis, Wegener's granulomatosis, acnevulgaris, Alzheimer's disease, autoimmune diseases, hypersensitivities,Parkinson's disease, etc., and combinations thereof.

The method can include systemic administration of the Nsp-IL10polypeptide or polynucleotide. Alternatively, the method can includelocal administration of the Nsp-IL10 polypeptide or polynucleotide. Forexample, the Nsp-IL10 polypeptide or polynucleotide can be administeredlocally to areas of inflammation such as inflamed joints. In someembodiments, the Nsp-IL10 polypeptide or polynucleotide is administeredto areas of the subject comprising chondrocytes.

In some embodiments, the method treats the disease by reducinginflammation, pain, tissue degeneration, or combinations thereof. Insome embodiments, the method reduces inflammation locally in areasaffected by osteoarthritis. In some embodiments, the method treats thedisease by activating an NGF signaling pathway, an IL10 signalingpathway, or combinations thereof. In some embodiments, the methodincreases phosphorylation of TrkA, increases expression of SIRT1,increases phosphorylation of cAMP response element-binding protein(CREB), or combinations thereof.

In some embodiments, the method includes treating a subject with adisease comprising administering to the subject a medicament comprisingan Nsp-IL10 polypeptide comprising an Nsp polypeptide and an IL10polypeptide. Generally, the medicament comprises a pharmaceuticallyacceptable excipient and a pharmaceutically effective amount of anNsp-IL10 polypeptide comprising an Nsp polypeptide and an IL10polypeptide.

Also disclosed herein are methods of activating an anti-inflammatorysignaling pathway in a biological cell comprising administering to thecell an Nsp-IL10 polypeptide comprising an Nsp polypeptide and an IL10polypeptide. The anti-inflammatory signaling pathway can comprise an NGFpathway or an IL10 pathway. In some embodiments, both an NGF pathway andan IL10 pathway are activated. In some embodiments, the biological cellis a human cell. In some embodiments, the cell is a chondrocyte.

Kits

Also disclosed herein are kits comprising a vector comprising apolynucleotide sequence encoding an Nsp-IL10 polynucleotide operablylinked to a promoter. The Nsp-10 polynucleotide comprises an Nsppolynucleotide and an IL10 polynucleotide.

In some embodiments, the NGF polypeptide or polynucleotide is thatidentified in one or more publicly available databases as follows: HGNC:7808, Entrez Gene: 4803, Ensembl: ENSG00000134259, OMIM: 162030, andUniProtKB: P01138. In some embodiments, the NGF polynucleotide comprisesthe sequence of SEQ ID NO: 8, or a polynucleotide sequence having at orgreater than about 80%, at or greater than about 85%, at or greater thanabout 90%, at or greater than about 95%, or at or greater than about 98%homology with SEQ ID NO: 8. The NGF polynucleotide can be from anyvertebrate, particularly from any mammal such as livestock such as cows,pigs, and sheep, primates such as humans, gorillas and monkeys, rodentssuch as mice, rats and guinea pigs, and other mammals such as horse,dog, bear, deer, dolphin, felines, etc. In some embodiments, the Nsppolynucleotide is a portion of human NGF.

The Nsp polynucleotide can encode more than one portion of NGF (e.g. anN-terminal portion and a C-terminal portion). In some embodiments, theNsp polynucleotide encodes the N-terminal half of NGF. Optionally, theNsp polynucleotide encodes the unstructured N-terminal domain of NGF. Insome embodiments, the Nsp polynucleotide encodes amino acids 1-14 ofNGF.

In some embodiments, the Nsp polynucleotide contains at least 60% (forexample, at least 60%, at least 65%, at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%) identity to SEQ IDNO: 9. In some embodiments, the Nsp polynucleotide comprises thesequence of SEQ ID NO: 9.

In some embodiments, the IL10 polynucleotide encodes a full-length IL10including a signal peptide. In other embodiments, the IL10polynucleotide encodes a form of IL10 lacking a signal peptide. In someembodiments, the IL10 polynucleotide is from any vertebrate,particularly from any mammal such as livestock such as cows, pigs, andsheep, primates such as humans, gorillas and monkeys, rodents such asmice, rats and guinea pigs, and other mammals such as horse, dog, bear,deer, dolphin, felines, etc.

In some embodiments, a polynucleotide encoding the IL10 polypeptidecomprises a nucleic acid sequence which is at least 60%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% identical to SEQ ID NO: 11. In some embodiments, apolynucleotide encoding the IL10 polypeptide comprises SEQ ID NO: 11.

In some embodiments, the Nsp-IL10 polynucleotide further comprises alinker. For example, the Nsp polynucleotide may be linked to the IL10polynucleotide by an intervening linker comprising one or morenucleotides. The linker can contain one, two, three, four, five, six,seven, eight, nine, ten, or a plurality of nucleotides. The Nsp-IL10polynucleotide can comprise more than one linkers (e.g., one, two,three, four, five, six, seven, eight, nine, ten, or a plurality oflinkers).

In some embodiments, a linker is between the Nsp polynucleotide and theIL10 polynucleotide. In some embodiments, the linker is positionedbetween a 5′ end of an Nsp polynucleotide and a 3′ end of a IL10polynucleotide. Alternatively, the linker is positioned between a 3′ endof an Nsp polynucleotide and a 5′ end of a IL10 polynucleotide. In someembodiments, the continuous Nsp polynucleotide is inserted within thesequence of the IL10 polynucleotide, wherein a linker is positionedbetween the Nsp polynucleotide and the IL10 polynucleotide. In someembodiments, the Nsp polynucleotide is inserted within the sequence ofthe IL10 polynucleotide 3′ to an IL10 signal peptide and 5′ to an IL10polynucleotide, wherein a linker is positioned between the Nsppolynucleotide and the IL10 polynucleotide. In some embodiments, thepolynucleotide comprises an Nsp polynucleotide flanked by two linkersinserted within the sequence of an IL10 polynucleotide.

In some embodiments, a polynucleotide encoding the linker comprises anucleic acid sequence which is at least 60%, at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%identical to SEQ ID NO: 13. In some embodiments, a polynucleotideencoding the linker comprises SEQ ID NO: 13.

In some embodiments, a polynucleotide encoding the Nsp-IL10 polypeptidecomprises a nucleic acid sequence which is at least 60%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% identical to SEQ ID NO: 14. In some embodiments, apolynucleotide encoding the Nsp-IL10 polypeptide comprises SEQ ID NO:14.

Non-limiting examples of vectors that can be used to introduceexpression vectors that encode Nsp-10 polypeptide in various cell types:a nucleic acid vector (e.g., a plasmid vector) encoding Nsp-10polypeptide can be delivered directly to bacterial cells or culturedcells (e.g., mammalian cells) by electroporation; a polynucleotidevector (e.g., a plasmid vector) encoding Nsp-10 polypeptide can bedelivered directly to bacterial cells by chemical transformation; aviral vector (e.g., a retroviral vector, adenoviral vector, an adenoassociated viral vector, an alphavirus vector, a vaccinia viral vector,a herpes viral vector, etc., as are known in the art) comprising apolynucleotide sequence encoding Nsp-10 polypeptide can be used todeliver Nsp-10 polypeptide to cells (e.g., mammalian cells); abaculovirus expression system can be used to deliver Nsp-10 polypeptideto insect cells; Agrobacterium mediated delivery can be employed inplants; and/or lipid mediated delivery (e.g., lipofectamine,oligofectamine) can also be employed for mammalian cells.

In some embodiments, the gene sequence (for example, of a geneexpressing Nsp-10 polypeptide) may be codon optimized, without changingthe resulting polypeptide sequence. In some embodiments, the codonoptimization includes replacing at least one, or more than one, or asignificant number, of codons with one or more codons that are morefrequently used in various organisms. In some embodiments, the codonoptimization increases expression of the optimized gene sequence.

EXAMPLES

To further illustrate the principles of the present disclosure, thefollowing examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompositions, articles, and methods claimed herein are made andevaluated. They are not intended to limit the scope of the presentinvention. These examples are not intended to exclude equivalents andvariations of the present invention which are apparent to one skilled inthe art. Unless indicated otherwise, temperature is ° C. or is atambient temperature, and pressure is at or near atmospheric. There arenumerous variations and combinations of process conditions that can beused to optimize product quality and performance. Only reasonable androutine experimentation will be required to optimize such processconditions.

Example 1. Development and Functional Analysis of Nsp-IL10 PolypeptideExpression System in Chondrocytes

The polypeptide Nsp-IL10 can be constructed in numerous ways. Severalembodiments of an Nsp-IL10 polypeptide containing the NGFR targeteddomain NGF Small Peptide (“Nsp”) inserted at the N- and/or C-terminus ofan IL10 polypeptide are shown in FIG. 1A. The expression constructs cancontain, for example, a Myc-DDK tag for purification and/oridentification purposes. A cytomegalovirus (CMV) promoter is an examplepromoter which can be used to drive expression of the polypeptide mRNA.Three example construct strategies (a, b, and c) are shown, representingdifferent placements of an Nsp sequence within the recombinantconstruct. Strategy a includes fusion of an Nsp sequence at theC-terminal end of an IL10 polypeptide. Strategy b, further detailed inFIG. 2, includes insertion of an Nsp sequence at the N-terminus of anIL10 polypeptide and at the C-terminal end of an IL10 signal peptide.Strategy c includes a combination of strategies a and b. Item d depictsthe human IL-10 vector control, in which no Nsp sequence is included.Internal ribosome entry sites (IRES) are included in each construct,which can drive expression of a reporter gene to track transcription ofthe overall construct. Any reporter gene capable of trackingtranscription can be used; for example, green fluorescent protein (Gfp).

The predicted protein structure of Nsp-IL10 construct b from RaptorXshows no conformational change in IL-10 in polypeptide expressionstrategy b (FIG. 1B). Amino acids 19-178 of IL-10 retain nativestructure, despite insertion of the Nsp sequence near the N-terminus ofIL-10.

Because Nsp specifically binds NGFR, Nsp functions to target Nsp-IL10polypeptide to NGFR for specific delivery of IL-10 to NGFR+ cells (FIG.1C). Nsp specifically binds the NGF receptor TrkA, thereby additionallypositioning IL-10 adjacent to NGFR+ cells. Thus, Nsp-IL10 polypeptide iscapable of activating both the NGFR and IL-10 signaling pathways, eitherin the same cell or in separate, adjacent or nearby cells.

Plasmid-based transgene constructs can be sub-cloned into viral vectors,which can be transduced into mammalian cells for efficient, heterologousexpression of proteins. An example construct used for Nsp-IL10polypeptide construction and expression in mammalian cells is shown inFIG. 2A. In one example embodiment, strategy b of FIG. 1A was used todevelop an Nsp-IL10 polypeptide and determine expression in human cells(FIG. 2B). The Nsp sequence was placed at the N-terminus of IL10polypeptide lacking a signal peptide and at the C-terminus of an IL10signal peptide in a human IL-10 expression construct. The constructfurther contained a Myc-DDK tag for purification and/or identificationpurposes. The gene construct was transduced into a HEK293 cell line andanalyzed for expression (FIG. 2B). IL-10 Western blot analysis ofsupernatants from cultures of HEK293 cells showed results from cellsharboring non-transgene control (Ctrl), human IL-10 transgene (hIL10)and Nsp-IL10 polypeptide transgene (Nsp-IL10), respectively.

The function of Nsp-IL10 protein from HEK293 culture medium wasanalyzed. Primary isolated human derived articular chondrocytes (AC)were used as reporter cells of NGF and IL-10 signaling. ACs wereisolated from healthy (“Healthy AC”) and diseased (“OA AC”) areas of thesame total knee joint replacement patient. ACs were treated withNsp-IL10-containing medium for 48 hours. Western blot analysis showedthat within 48 hours Nsp-IL10 treatment activated the NGF receptorTyrosine kinase membrane receptor A (TrkA), as shown by appearance ofphosphorylated TrkA (p-TrkA). Further, expression of a marker generelated to the control of cellular aging, Sirtuin 1 (SIRT1) was enhancedwith IL-10 treatment compared to untreated controls (Ctrl). SIRT1inhibits apoptosis and enhances survival of human OA chondrocytes.Results of p-TrkA and SIRT1 Western blot analysis indicated simultaneousactivation of both NGF and IL10 mediated signaling pathways. P2,DMEM:F12 1:1, 10% fetal bovine serum (FBS) was used as the HEK293 cellculture conditions. Cultures were grown to full confluence, thenpre-incubated in 2% FBS hDMEM for 24 hours. Cells were then removed bycentrifugation, and culture medium was added to AC cultures for 48hours. Anti-GAPDH antibody was used as a loading control in Western blotexperiments.

REFERENCES

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Example 2. Sequences

An NGF amino acid sequence SEQ ID NO: 1MSMLFYTLITAFLIGIQAEPHSESNVPAGHTIPQAHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRK AVRRAAn Nsp amino acid sequence SEQ ID NO: 2 SSSHPIFHRGEFSVAn IL-10 amino acid sequence. SEQ ID NO: 3MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIR NAn IL-10 amino acid sequence SEQ ID NO: 4SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN An IL-10 amino acid sequenceSEQ ID NO: 5 MHSSALLCCLVLLTGVRA A linker amino acid sequenceSEQ ID NO: 6 GGSG An Nsp-IL10 polypeptide amino acid sequenceSEQ ID NO: 7MHSSALLCCLVLLTGVRAGGSGSSSHPIFHRGEFSVGGSGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN A DNA sequence encoding the NGF polypeptide ofSEQ ID NO: 1 SEQ ID NO: 8ATGTCCATGTTGTTCTACACTCTGATCACAGCTTTTCTGATCGGCATACAGGCGGAACCACACTCAGAGAGCAATGTCCCTGCAGGACACACCATCCCCCAAGCCCACTGGACTAAACTTCAGCATTCCCTTGACACTGCCCTTCGCAGAGCCCGCAGCGCCCCGGCAGCGGCGATAGCTGCACGCGTGGCGGGGCAGACCCGCAACATTACTGTGGACCCCAGGCTGTTTAAAAAGCGGCGACTCCGTTCACCCCGTGTGCTGTTTAGCACCCAGCCTCCCCGTGAAGCTGCAGACACTCAGGATCTGGACTTCGAGGTCGGTGGTGCTGCCCCCTTCAACAGGACTCACAGGAGCAAGCGGTCATCATCCCATCCCATCTTCCACAGGGGCGAATTCTCGGTGTGTGACAGTGTCAGCGTGTGGGTTGGGGATAAGACCACCGCCACAGACATCAAGGGCAAGGAGGTGATGGTGTTGGGAGAGGTGAACATTAACAACAGTGTATTCAAACAGTACTTTTTTGAGACCAAGTGCCGGGACCCAAATCCCGTTGACAGCGGGTGCCGGGGCATTGACTCAAAGCACTGGAACTCATATTGTACCACGACTCACACCTTTGTCAAGGCGCTGACCATGGATGGCAAGCAGGCTGCCTGGCGGTTTATCCGGATAGATACGGCCTGTGTGTGTGTGCTCAGCAGGAAGGCTGTGAGAAGAGCCTGAA DNA sequence encoding the Nsp polypeptide of SEQ ID NO: 2 SEQ ID NO: 9TCATCATCCCATCCCATCTTCCACAGGGGCGAATTCTCGGTGA DNA sequence encoding the IL-10 polypeptide of SEQ ID NO: 3SEQ ID NO: 10 ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGAA DNA sequence encoding the IL10 amino acid sequence of SEQ ID NO: 4SEQ ID NO: 11 AGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGAA DNA sequence encoding the IL10 amino acid sequence of SEQ ID NO: 5SEQ ID NO: 12 ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCA DNA sequence encoding the linker amino acid sequence of SEQ ID NO: 6SEQ ID NO: 13 GGAGGATCAGGCA DNA sequence encoding the Nsp-IL10 polypeptide of SEQ ID NO: 7SEQ ID NO: 14 ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCGGAGGATCAGGCTCATCATCCCATCCCATCTTCCACAGGGGCGAATTCTCGGTGGGAGGATCAGGCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGA

Publications cited herein are hereby specifically incorporated byreference in their entireties and at least for the material for whichthey are cited.

It should be understood that, while the present disclosure has beenprovided in detail with respect to certain illustrative and specificaspects thereof, it should not be considered limited to such, asnumerous modifications are possible without departing from the broadspirit and scope of the present disclosure as defined in the appendedclaims. It is, therefore, intended that the appended claims cover allsuch equivalent variations as fall within the true spirit and scope ofthe invention.

We claim:
 1. A method of treating joint inflammation in a subjectcomprising administering to the subject a therapeutically effectiveamount of an Nsp-IL10 polypeptide comprising an Nsp polypeptide and anIL10 polypeptide, wherein the Nsp polypeptide is at least 90% identicalto the full length of SEQ ID NO: 2, and wherein the administrationresults in a reduction of the joint inflammation in the subject.
 2. Themethod of claim 1, wherein the Nsp polypeptide consists of SEQ ID NO: 2.3. The method of claim 1, wherein the IL10 polypeptide comprises SEQ IDNO:
 4. 4. The method of claim 1, wherein the joint inflammation ischronic.
 5. The method of claim 1, wherein the joint inflammationcomprises osteoarthritis.
 6. The method of claim 1, wherein theadministration of the Nsp-IL10 polynucleotide results in activation ofan NGF signaling pathway and an IL-10 signaling pathway.
 7. The methodof claim 1, wherein the method increases phosphorylation of TrkA,increases expression of SIRT1, increases phosphorylation of CREB, orcombinations thereof.
 8. The method of claim 1, wherein the Nsp-IL10polypeptide further comprises a signal peptide having a sequence of SEQID NO:
 5. 9. The method of claim 1, wherein the Nsp-IL10 polypeptidefurther comprises one or more linkers.
 10. The method of claim 9,wherein the one or more linkers is between the Nsp polypeptide and theIL10 polypeptide.
 11. The method of claim 9, wherein the one or morelinkers comprises SEQ ID NO:
 6. 12. The method of claim 1, wherein theNsp-IL10 polypeptide comprises SEQ ID NO:
 7. 13. The method of claim 1,wherein the method further treats tissue degeneration and theadministration further results in a reduction of tissue degeneration inthe subject.